Dm5000b

After the stimulation assay, epithelial and stromal cells were washed three times in sterile PBS, fixed for 10 min in 4% paraformaldehyde (Sigma), washed two times in PBS- 0.1% Triton for 5 min and incubated with the blocking solution (PBS + 2.5% BSA) for 1 h at room temperature. Cells were incubated overnight at 4 °C with rabbit polyclonal anti-IRF3 antibody (1:50, ab25950, Abcam^®, Cambridge, UK) and rabbit polyclonal anti-NFkB p65 antibody (1:100, sc-109-G, Santa Cruz biotechnology^®, Texas, USA). After washing twice for 10 min in PBS, the secondary antibody (1:300, Alex Fluor^® 594 Goat Anti-Rabbit IgG (H + L), Life Technologies, Carlsbad, USA) was added and cells incubated for 1 h at room temperature. After washing for 10 min in PBS, the glass coverslips were mounted in Vectashield^® mounting medium with DAPI (H-1200, Vector Laboratories Inc., Burlingame, CA, USA). Slides were observed using a fluorescence microscope (Leica DM5000B) and the images obtained in Adobe Photoshop CS5.

To analyse the subcellular localization of the Cas9-GFP fused protein, the hyphae cultivation and microscopic analysis were performed as described in Wanka et al. [19 (link)]. Briefly, two disinfected coverslips were placed onto the bottom of a small petri dish, and then 5 mL of liquid MM was supplemented with 0.003% yeast extract. After inoculation with 10⁶ spores of A. niger, the petri dishes were incubated for 8 h at 30 °C. After incubated with 4′, 6-diamidino-2-phenylindole (DAPI) at the final concentration of 1 mg/mL for 15 min, coverslips with adherent hyphae were placed upside down on an object slide and analysed by microscopy. Differential interference contrast (DIC) and green fluorescent images of the cells were captured with a 40× objective using a Leica DM5000B and the results were assembled in Adobe Photoshop 7.0 (Adobe, San Jose, CA).